In prior art imaging systems, the image of an object gradually goes out of focus as the object moves from the plane of best focus. The image of a portion of the object that is in focus is also degraded by the image of adjacent portions of the object that are out of focus. This effect causes certain problems when detailed information is desired from the in-focus portion of the object and without influence from the surrounding portions of the object. It is particularly important in the field of microscopy to avoid degradation of the in-focus image due to adjacent out of focus images.
Previous methods of obtaining a clear image of the desired portion or plane of an object include the use of pupil-plane filters. Pupil-plane filters utilize either amplitude (absorption) modulation or phase modulation of the light distribution in the pupil plane. Continuously varying amplitude pupil plates and annular binary pupil plates have been used to reduce the width of the central lobe of the axial intensity point spread function (PSF). These amplitude plate-based methods share two serious drawbacks: decreased optical power at the image plane and possible decrease in the lateral image resolution. A phase-only pupil filter has also been used to reduce the axial spot size of a confocal scanning microscope. However, such a filter is not applicable to a hybrid imaging system because it employs a phase filter to reduce the width of the axial main lobe. However, due to the extremely high side-lobes in the PSF of such a phase filter, the useful optical power is reduced significantly.
Structured illumination is another prior art approach to reducing the depth of field of an imaging system. For example, M. Neil et al., Method of obtaining optical sectioning by using structured light in a conventional microscope, Optics Letters, vol. 22, no. 24, pp. 1905–1907 (1997), demonstrated that sinusoidal fringes of light would be formed by interference and projected onto the object. When an image is formed, the fringes go out of focus faster than a normal image. This effect leads to a slightly smaller depth of field; but the portions of the image that lie in the nulls of the sinusoidal fringes are lost. By the use of multiple exposures where the sinusoidal fringe is moved by a fraction of the fringe period for additional images, the complete image of the object can be retrieved when all of the images are superimposed on one another. One disadvantage of structured illumination is that precise alignment is needed. Another disadvantage is that multiple exposures must be made of the object to obtain a single image. This necessity for multiple exposures is problematic, especially when the object is moving, as in the case of live objects or moving parts along an assembly line. In the case of fluorescence microscopy, since the fluorophore is being bleached by the ultraviolet light used to excite the fluorophore, the later images are dimmer. If the object is a live cell, the ultraviolet light also damages the cell, making additional exposures particularly harmful.
In confocal microscopy, optical “slices” are produced by focusing a point source onto the specimen and by imaging that point onto a point detector. Out-of-focus light is preferably removed to produce an in-focus image. Unfortunately, in order to obtain a complete image of the specimen, each plane of the specimen must be scanned point by point and the images of each plane then combined to achieve a three-dimensional result. Therefore, confocal microscopy is time consuming and is not suitable for imaging rapidly changing objects—such as living or moving samples.
In contrast, the deconvolution microscope works with images of slices taken by a standard imaging system. Once images of the slices are taken and stored, along with the in-focus and out-of-focus PSFs of the imaging system, the deconvolution microscope attempts to calculate the image in each plane. However, the digital post-processing used in calculating the image generates many undesirable artifacts.